Category. Antiretroviral (Nucleoside/Nucleotide Reverse Transcriptase Inhibitor).

Storage. Lamivudine oral solution should be kept in a well-closed container.

Additional information. Strength in the current WHO Model List of Essential Medicines (EML): 50 mg per 5 mL (10 mg per mL). Strength in the current WHO EML for children: 50 mg per 5 mL (10 mg per mL).

Requirements

Comply with the monograph for "Liquid preparations for oral use".

Definition. Lamivudine oral solution is a solution of Lamivudine in a suitable vehicle which may be flavoured. It contains not less than 90.0% and not more than 110.0% of the amount of Lamivudine (C₈H₁₁N₃O₃S) as stated on the label.

Identity tests

• Any two of tests A, B or C may be applied.

1. Carry out the test as described under 1.14.1 Chromatography, Thin-layer chromatography, using silica gel R6 as the coating substance and a mixture of 67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol R and 3 volumes of ammonia (~260 g/L) TS as the mobile phase. Apply separately to the plate 10 μL of each of the following two solutions. For solution (A), dilute 5 mL of the oral solution, nominally containing 50 mg of Lamivudine, to 50 mL with methanol R, filter, and use the filtrate. For solution (B), use a solution containing 1.0 mg of lamivudine RS per mL of methanol R. After removing the plate from the chromatographic chamber, allow it to dry in a current of air and examine the chromatogram in ultraviolet light (254 nm). The principal spot obtained with solution (A) corresponds in position, appearance and intensity to that obtained with solution (B).

   Spray the plate with vanillin/sulfuric acid TS1. Heat the plate for a few minutes at 120°C. Examine the chromatogram in daylight. The principal spot obtained with solution (A) corresponds in position, appearance and intensity to that obtained with solution (B).

2. The absorption spectrum (1.6) of solution (1), when observed between 210 nm and 300 nm, exhibits one maximum at about 280 nm.

   Alternatively, in combination with identity test C, where a diode array detector is available, record the UV spectra of the principal peaks in the chromatograms with a diode array detector in the range of 210 nm to 300 nm. The UV spectrum of the principal peak in the chromatogram obtained with solution (1) corresponds to the UV spectrum of the peak due to lamivudine in the chromatogram obtained with solution (2).

3. Carry out the test as described under 1.14.1 Chromatography, High-performance liquid chromatography, using the conditions and solutions given under "Assay". The retention time of the principal peak in the chromatogram obtained with solution (1) corresponds to the retention time of the peak due to lamivudine in the chromatogram obtained with solution (2).

pH value (1.13). pH of the oral solution, 5.0 – 7.0.

Related substances. Carry out the test as described under 1.14.1 Chromatography, High-performance liquid chromatography, using a stainless steel column (25 cm x 4.6 mm) packed with end-capped particles of silica gel, the surface of which has been modified with chemically-bonded octylsilyl groups (5 µm).

Use the following conditions for gradient elution:

• Mobile phase A: dissolve 1.0 g sodium octanesulfonate R in 1000.0 mL of water R, add 5.0 mL of phosphoric acid (~680 g/L) TS and mix.
• Mobile phase B: 25 volumes of methanol R and 75 volumes of acetonitrile R.

Operate with a flow rate of 1.5 mL per minute. As a detector, use an ultraviolet spectrophotometer set at a wavelength of 277 nm. Maintain the column temperature at 40 °C.

Prepare the following solutions using as a diluent a mixture of 80 volumes of water R and 20 volumes of methanol R. For solution (1), transfer a volume of the oral solution, nominally containing 50 mg of Lamivudine, to a 100 mL volumetric flask. Add about 60 mL of diluent and sonicate for about 20 minutes with intermittent shaking. Allow to cool to room temperature, dilute to volume and filter. For solution (2), dilute 1.0 mL of solution (1) to 100.0 mL. For solution (3), dilute 5.0 mL of solution (2) to 100.0 mL. For solution (4), dissolve 5 mg of lamivudine for system suitability 1 RS (containing lamivudine and lamivudine impurities A and B) and dilute to 10.0 mL. For solution (5), dissolve 25 mg of cytosine R, 25 mg of uracil R and 25 mg of salicylic acid R and dilute to 50.0 mL. Dilute 1.0 mL of this solution to 100.0 mL. For solution (6), dissolve a suitable amount of each of the excipients stated on the label in 10 mL of a suitable solvent and dilute to 100.0 mL with the diluent.

Inject 20 μL each of solutions (1), (2), (3), (4), (5) and (6).

The test is not valid unless, in the chromatogram obtained with solution (4), the resolution factor between the peaks due to lamivudine and impurity B is at least 5.0. Also, the signal-to-noise ratio of the peak due to lamivudine in the chromatogram obtained with solution (3) is at least 10.

Use the chromatogram obtained with solutions (4) and (5) to identify peaks due to impurities in the chromatogram obtained with solution (1), if present. The impurity peaks are eluted at the following relative retention with reference to lamivudine (retention time about 32 minutes): impurity F about 0.08; impurity J about 0.12; impurity E about 0.27; impurity G about 0.30; impurity H about 0.31; impurity A about 0.63; impurity B about 0.84. Use the chromatogram obtained with solution (6) to identify the peaks due to excipients.

In the chromatogram obtained with solution (1):

• the area of any peak corresponding to impurity J, when multiplied by a correction factor of 2.2, is not greater than 1.5 times the area of the peak due to lamivudine in the chromatogram obtained with solution (2) (1.5%);

• the area of any peak corresponding to impurity H is not greater than 0.6 times the area of the peak due to lamivudine in the chromatogram obtained with solution (2) (0.6%);

• the area of any peak corresponding to impurity E, when multiplied by a correction factor of 0.6, is not greater than 0.3 times the area of the peak due to lamivudine in the chromatogram obtained with solution (2) (0.3%);

• the area of any peak corresponding to impurity G is not greater than 0.3 times the area of the peak due to lamivudine in the chromatogram obtained with solution (2) (0.3%);

• the area of any other impurity peak is not greater than 0.2 times the area of the peak due to lamivudine in the chromatogram obtained with solution (2) (0.2%).

• The sum of the areas of all impurity peaks, including the corrected areas of any peaks corresponding to impurities J and E and the areas of all other impurity peaks, is not greater than two times the area of the peak due to lamivudine obtained with solution (2) (2.0%). Disregard any peaks with an area less than the area of the principal peak obtained with solution (3) (0.05%) and with the same retention times as any of the peaks in the chromatogram obtained with solution (6).

Assay. Carry out the test as described under 1.14.1 Chromatography, High-performance liquid chromatography, using a stainless steel column (25 cm x 4.6 mm) packed with end-capped particles of silica gel, the surface of which has been modified with chemically bonded octadecylsilyl groups (5 μm).

Use the following conditions for gradient elution:

• Mobile phase A: dissolve 1.0 g sodium octanesulfonate R in 1000.0 mL of water R, add 5.0 mL of phosphoric acid (~680 g/L) TS and mix.
• Mobile phase B: acetonitrile R.

Operate with a flow rate of 1.5 mL per minute. As a detector, use an ultraviolet spectrophotometer set at a wavelength of 255 nm. Maintain the column temperature at 30 °C.

Prepare the following solutions using as a diluent a mixture of 80 volumes of water R and 20 volumes of methanol R. For solution (1), transfer a volume of the oral solution, nominally containing 100 mg of Lamivudine, to a 200 mL volumetric flask. Add 120 mL of diluent and sonicate for about 20 minutes with frequent shaking. Allow to cool to room temperature, dilute to volume and filter. For solution (2), dissolve 50.0 mg of lamivudine RS and dilute to 100.0 mL.

Inject alternately 20 μL each of solutions (1) and (2).

Measure the areas of the peaks corresponding to lamivudine in the chromatograms of solutions (1) and (2) and calculate the percentage content of lamivudine (C₈H₁₁N₃O₃S), weight in volume, in the oral solution, using the declared content of (C₈H₁₁N₃O₃S) in lamivudine RS.

Impurities. The impurities limited by the requirements of this monograph include those listed in the monograph for Lamivudine (excluding impurity D).